The authors would like to acknowledge the Interdisciplinary Innovation Initiative Program, University of Illinois, grant 12035. S.K. was funded at UIUC from National Science Foundation (NSF) Grant 0965918 IGERT: Training the Next Generation of Researchers in Cellular and Molecular Mechanics and BioNanotechnology.&#160;This work was supported in part by the grant from the nanoBIO node of the National Science Foundation ( ECC-1227034).

Fabricating and functionalizing PA gels of varying stiffnesses with fluorescent microspheres embedded near the cell culture surface.
1. Functionalizing the Top Glass Cover Slips
<ol>
	<li>Clean glass cover slips (#1.0, 12 mm diam.) with soap and water, followed by ethanol to remove extraneous dust.</li>
	<li>Place glass cover slips on a grated surface (i.e. pipette tip holder) such that they are not touching to facilitate ease of interaction with the coverslips.</li>
	<li>Coat the e...

<ol>
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X., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+deeply+cells+feel:+methods+for+thin+gels.">How deeply cells feel: methods for thin gels.</a> J. Phys.: Condens. Matter. 22 (2010), 194116-194126 (2010).</li><li>Polio, S. R., Rothenberg, K. E., Stamenovic, M. L., Smith, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+micropatterning+and+image+processing+approach+to+simplify+measurement+of+cellular+traction+forces.">A micropatterning and image processing approach to simplify measurement of cellular traction forces.</a> Acta Biomaterialia. 8, 82-88 (2012).</li><li>Wang, Y. L., Pelham, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+a+flexible,+porous+polyacrylamide+substrate+for+mechanical+studies+of+cultured+cells.">Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells.</a> Methods in Enzymol. 298, 489-496 (1998).</li><li>Tse, J. R., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Hydrogel+Substrates+with+Tunable+Mechanical+Properties.">Preparation of Hydrogel Substrates with Tunable Mechanical Properties.</a> Current Protocols in Cell Biol. , 16 (2010).</li><li>Poellmann, M. J., Wagoner Johnson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+and+Patterning+Polyacrylamide+Substrates+Functionalized+with+N-Hydroxysuccinimide.">Characterizing and Patterning Polyacrylamide Substrates Functionalized with N-Hydroxysuccinimide.</a> Cell and Mol. Bioengineering. 6 (3), 299-309 (2013).</li><li>Tseng, Q., Duchemin-Pelletier, E., Deshiere, A., Balland, M., Guillou, H., Filhol, O., Th&#233;ry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+organization+of+the+extracellular+matrix+regulates+cell&#8211;cell+junction+positioning.">Spatial organization of the extracellular matrix regulates cell&#8211;cell junction positioning.</a> PNAS. 109 (5), 1506-1511 (2012).</li><li>Trappmann, B., Gautrot, J. E., Connelly, J. T., Strange, D. G. T., Li, Y., Oyen, M. L., Cohen Stuart, M. A., Boehm, H., Li, B., Vogel, V., Spatz, J. P., Watt, F. M., Huck, W. T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular-matrix+tethering+regulates+stem-cell+fate.">Extracellular-matrix tethering regulates stem-cell fate.</a> Nature Materials. 11, 642-649 (2012).</li><li>Wong, J. Y., Leach, J. B., Brown, X. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balance+of+chemistry,+topography,+and+mechanics+at+the+cell-biomaterial+interface:+Issues+and+challenges+for+assessing+the+role+of+substrate+mechanics+on+cell+response.">Balance of chemistry, topography, and mechanics at the cell-biomaterial interface: Issues and challenges for assessing the role of substrate mechanics on cell response.</a> Surface Science. 570 (1-2), 119-133 (2004).</li><li>Mih, J. D., Sharif, A. 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P., Chen, J., Wang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+traction+response+in+human+airway+smooth+muscle+cells.">Spatial and temporal traction response in human airway smooth muscle cells.</a> Am J Physiol Cell Physiol. 283, 1254-1266 (2002).</li><li>Atanackovic, T., Guran, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Theory of Elasticity for Scientists and Engineers. , (2000).</li></ol>

The authors declare that they have no competing financial interests.

When using this technique, it is essential that the bead solution is properly diluted and the beads are chosen based on desired diameter, PA gel substrate stiffness, and the size scale of the phenomena that is explored in the desired experiment.
Caution should be taken when diluting the bead solution prior to functionalizing top glass cover slips. The spacing between beads on the gel surface can be altered by changing the dilution factor of the colloid solution. Diluting the solution too much ...

The mechanical interaction of a living cell with its local environment has commonly been studied using PA gels. These substrates rely on a simple, well-characterized protocol established by Dembo and Wang in 19971. One of the main advantages of these substrates is that their stiffness may be tuned by modifying the concentrations of specific components of the gel solution. This provides a desirable platform to study cells&#8217; interaction with environments of different rigidities. When PA gels are coated with extracellular matrix (ECM) proteins, cells adhere to them, generating force. As a result of cell force, the gel deforms as an elastic body. This deformation depends on the magnitude of the force applied by the cells and the elastic properties of the gel. Various studies have employed PA gels to investigate cellular traction forces.
In one variation of PA gel fabrication, fluorescent microspheres (beads) are embedded throughout the gel to quantify cell traction forces on gels of different rigidities2. Upon cell force application, the beads displace from their initial location following gel deformation. The deformation field is measured from the individual bead displacements. This deformation field is utilized with elasticity theory and the elastic properties of the gel to compute the traction forces. These measurements provide insight as to how cells mechanically sense and interact with their local microenvironment3.
In many widely used PA gel fabrication protocols, the beads are intermixed throughout the PA gel in its liquid, unpolymerized state. A fully polymerized PA gel contains fluorescent beads throughout its volume. When computing cell traction forces, beads most near the gel surface (cell-substrate interface) are monitored. The displacements of these beads are assumed to occur on the cell culture surface for simplicity in the force calculation. The actual location of the beads within the depth of the gel is disregarded. However, in an elastic medium (such as PA gel), a bead closer to a point of force application will move more than a bead that is further away from the point. Thus, treating the displacement of a point (at the bead location) distal from the surface as that at the surface results in an underestimation of cellular tractions. The degree of error depends on the distance of the bead from the surface. The error cannot be estimated without the knowledge of the location of the bead.
The need for a simple method to confine beads very near the cell culture surface has been addressed by a few techniques. One way is to increase the density of beads throughout the whole gel such that there are a sufficient number of beads in the top focal plane to measure motion very near the surface. Another technique involves building a confocal imaging chamber for live cell imaging such that the light from only the beads in the upper-most focal plane is collected4. A different method involves overlaying an extremely thin layer of PA gel containing beads on top of an already polymerized gel without beads5. A drawback of each of these techniques is that the precise location of the beads within the gel is not known. This introduces error into the calculation of the displacement field of the beads, and thus the calculation of cell forces. Another technique involves conjugation of beads to the top surface of an already polymerized PA gel using Sulfo-SANPAH6. This technique ensures the beads are indeed only on the top of the PA gel, but the extent to which they are embedded in the depth of the gel is unknown. This could potentially create a local topography for the cells, which could alter cell behavior, as prior work has suggested that cells can sense force several microns away7. Recently, a technique for patterning PA gels with 1 &#956;m diameter fluorescent fibronectin dot markers in a regularized array was established8. In this case, the depth of the fluorescent markers is known, and is essentially zero, as the fibronectin pattern is indirectly printed on the gel surface. However, this method does not provide a continuous environment on which cells can attach, as the ECM protein is limited to 1 &#956;m diameter dots. A method for fully integrating tracker beads within PA gels and confining them to a known location very near the surface has yet to be established.
Here, we develop a technique to constrain sub-&#956;m to &#956;m diameter fluorescent beads to a focal plane very near the cell culture surface within PA gel. A gel is typically cured by sandwiching unpolymerized liquid gel solution between two glass plates. One of the plates is functionalized so that the gel strongly adheres to it. The other is unfunctionalized and is removed after the gel cures. We modify this removable glass surface by coating it with a layer of beads. Upon sandwiching the liquid gel between the functionalized and the bead coated glass surface, the beads transfer to the gel while it is curing. This limits the distance of the beads&#8217; integration into the gel within 1.6 &#181;m of the surface. Glass-bottom Petri dishes are used as the adherent surface on which the gel is cured. To form a flat top gel surface during polymerization, a circular glass cover slip is used to sandwich the gel with the glass-bottom Petri dish. Prior to gel fabrication, the top glass cover slip is coated with poly-D-lysine (PDL), yielding a positive surface charge. The PDL is blown off with compressed air, and a solution of beads in water is deposited on the cover slips. We utilize carboxylated fluorescent microbeads, which carry a negative charge, and interact with the positively charged surface created by treatment with PDL. After blowing the bead solution off the coverslip with compressed air, a single layer of beads remains electrostatically coupled to the dry cover slip. The PDL coating does not affect the adhesiveness of the glass to the gel surface, as the glass slides are undamaged and removed from the PA gel fully intact.
The glass-bottom Petri dishes are made adherent by treatment with 97% 3-aminopropyl-trimethoxysliane and 0.5% glutaraldehyde. PA gels of desired rigidities are created by mixing appropriate concentrations of bisacrylamide and acrylamide via a standard procedure9. A droplet of the gel solution is pipetted onto the glass-bottom Petri dish. The glass cover slip containing the beads is used to sandwich the gel with the Petri dish. When the gel is cured, the top cover slip is removed leaving the beads embedded in the PA gel within 1.6 &#181;m from the surface.

<table border="0" cellpadding="0" cellspacing="0" width="502">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Name</td>
			<td style="width:128px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Reagents</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">97% 3-Aminopropyl-trimethoxysliane (APTES)</td>
			<td>Sigma-Aldrich</td>
			<td>281778</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">70% Glutaraldehyde</td>
			<td>Polysciences, Inc.</td>
			<td>111-30-8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">1M HEPES buffer solution</td>
			<td>Sigma-Aldrich</td>
			<td>83264</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">40% Acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A4058</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">2% Bisacrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>M1533</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">0.1 um Fluorescent microspheres (mCherry)</td>
			<td>Invitrogen</td>
			<td>F8801</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Fibronectin, Human, 1mg</td>
			<td>BD Biosciences</td>
			<td>354008</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ammonium Persulfate</td>
			<td>Bio-RAD</td>
			<td>161-0700</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Tetramethylethylenediamine (TEMED)</td>
			<td>Bio-RAD</td>
			<td>161-0801</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Poly-D-Lysine</td>
			<td>Millipore</td>
			<td>A-003-E</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)&#160;</td>
			<td>Thermo Scientific</td>
			<td>22980</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">N-hydroxysulfosuccinimide (NHS)</td>
			<td>Thermo Scientific</td>
			<td>24500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">.05% Trypsin-EDTA (1X)</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">NaCL</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Acrylic Acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>147230</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G7757</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">2-(N-morpholino)ethanesulfonic acid (MES)</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Materials</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">35 mm Glass bottom dish with 14 mm micro-well #1 cover glass</td>
			<td>In Vitro Scientific</td>
			<td>D35-14-1-N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glass cover slips, 12 mm diam.</td>
			<td>Ted Pella, Inc.</td>
			<td>26023</td>
		</tr>
	</tbody>
</table>

a novel method for localizing reporter fluorescent beads near the cell culture surface for traction force microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Confocal imaging was used to determine that the beads were indeed underneath the gel surface and to quantify their precise location within the gel depth. Fluorescent beads of a different wavelength than those inside the gel were allowed to settle on the surface, and the distance between the embedded fluorescent nanoparticles and those on the surface was calculated using a centroid identification algorithm. The location of the bead on the gel surface serves as a reference for the top surface of the gel&#8212;where cells a...

Watch this Scientific Journal Video about A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy at JoVE.com

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

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Research

JoVE Journal

Bioengineering

13.5K 조회수.  University of Illinois at Urbana-Champaign.  형광 프로브를 함유하는 폴리 아크릴 아미드 (PA) 겔을 제조하기위한 전통적인 기술은 접착 표면 및 유리 슬라이드 사이에 겔을 개재 포함한다. 여기서, 우리는 폴리-D-라이신 (PDL) 및 형광 프로브이 슬라이드 코팅은 겔 표면으로부터 1.6 μM 내로 프로브 지역화 것을 보여준다. 